Poster abstracts
Poster number 126 submitted by Michael Wolfe
Bru-Seq reveals modulation of mRNA stability by Human Pumilio proteins
Michael Wolfe, Peter Freddolino (Department of Biological Chemistry, University of Michigan, Department of Computational Medicine and Bioinformatics, University of Michigan), Trista Schagat (Department of Biological Chemistry, University of Michigan), Michelle Paulsen, Mats Ljungman (Department of Radiation Oncology, University of Michigan), Brian Magnuson (Department of Radiation Oncology, University of Michigan), Daeyoon Park, Aaron Goldstrohm (Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota), Chi Zhang (Department of Biological Sciences, University of Texas at Dallas)
Abstract:
Post-transcriptional gene regulation in eukaryotic systems is widely controlled through the binding of diverse regulators, including miRNAs and proteins, to the 3’ untranslated region of target mRNA molecules.1-3 The RNA binding proteins Human Pumilio1 (PUM1) and Pumilio 2 (PUM2) have been demonstrated to negatively regulate the protein expression of mRNA targets through binding of the consensus sequence 5’-UGUANAUW-3’.3-6 Previous studies in the Goldstrohm lab using RNA-seq have revealed nearly 1000 mRNA targets that either increase or decrease in expression when both PUM1 and PUM2 are knocked down with RNAi.7 However, RNA-seq is only able to assess the steady state levels of mRNA in any given condition and thus PUM effect on mRNA stability cannot be directly assessed. In order to separate the effects of mRNA stability from transcriptional activation, we employ a combination of Bromouridine sequencing (Bru-Seq) and Bromouridine-Chase sequencing (BruChase-Seq), in both WT and PUM knockdown HEK293 cells.8 Here, we reveal a total of 308 genes that have a statistically significant change in mRNA stability upon PUM knockdown. Of these genes, 252 increased in mRNA stability and 56 decreased in mRNA stability. On the contrary, only one gene was shown to have a significant change in transcription rate upon PUM KD. Motif analysis showed high mutual information between the UGUANAUW motif and genes that increased in mRNA stability upon PUM KD, suggesting that the primary mode of PUM mediated repression of gene expression is through the modulation of mRNA stability.
References:
References:
1). Mitchell et al. Nat Struct Mol Biol. Jan 2013; 20(1): 127–133.
2) Lee et al. Science. 2001 Oct 26;294(5543):862- 4.
3) Quenault et. al. Trends Cell Biol. 2011 Feb;21(2):104-12.
4) Wickens et al. Trends Genet. 2002 Mar;18(3):150-7.
5) Spassov et al. Gene. 2002 Oct 16;299(1-2):195-204
6) Galgano et. al. PLoS ONE. 2008; 3(9): e3164.
7) Bohn et al. (submitted, under review)
8) Paulsen, M. T. et al. Methods 67, 45–54 (2014).
Keywords: Bru-seq, mRNA stability , RBPs